Inhibition of nucleic acid polymerases by endonuclease V-cleavable circular oligonucleotide ligands

ABSTRACT

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. The oligonucleotide aptamers of the present invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 62/595,453, filed Dec. 6, 2017, the disclosure of whichis herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “0102384-002US0 SequenceListing.txt,” which was created on Dec. 5, 2017, and is 9 KB in size,are hereby incorporated by reference in their entirety.

BACKGROUND

Aspects of the present invention relate generally to improved methods ofblocking DNA polymerase activity with oligonucleotide aptamers at lowreaction temperatures, and restoring the enzyme activity upon raisingthe reaction temperature (e.g., hot-start methods).

DNA polymerases are enzymes used for synthesis of DNA strands by primerextension, wherein the polymerase-catalyzed DNA synthesis may beinitiated by oligonucleotide primers hybridized to a complementarytemplate DNA. Initiating DNA synthesis from this template-hybridizedprimer, DNA polymerases create complementary DNA strands in the presenceof corresponding nucleotide 5′-triphosphates. Sequence specificity ofnucleotide polymerization, when the oligonucleotide primers bindexclusively to the desired sites and nowhere else, is an importantrequirement in many applications wherein DNA synthesis is used. However,the efficiency and fidelity of DNA synthesis can be reduced when primershybridize to non-complementary DNAs, leading to synthesis of incorrectDNA sequences.

Many so-called ‘Hot Start’ methods have been developed to avoidincorrect primer extension products (e.g., see Paul N., et al. (2010),for review). One of the most common techniques is based on use ofoligonucleotide aptamers (Jayasena, S. D., 1999). Aptamers offer anumber of advantages over other reported methods. Using a method ofmolecular evolution (SELEX), they can be quickly engineered in a testtube and then readily and inexpensively manufactured by chemicalsynthesis. Ideally, an aptamer should: (i) completely block DNApolymerase at low temperatures, and (ii) provide no blockage effect atthe desired elevated reaction temperature. Unfortunately, this is verydifficult, if not impossible to achieve, and the aptamer structureusually represents a compromise between these two key requirements. Newmethods, therefore, are needed to improve control of aptamer activity inreaction mixtures containing DNA polymerases.

Particular aspects provide methods of activating an aptamer-inactivatedDNA polymerase, comprising: providing a reaction mixture suitable forDNA synthesis, the reaction mixture comprising (i) a DNA polymerase,(ii) an endonuclease V-cleavable circular oligonucleotide aptamer thatbinds to the DNA polymerase, wherein the oligonucleotide aptamer ispresent in an amount effective to inhibit DNA synthesis activity of theDNA polymerase in the reaction mixture, and (iii) an endonuclease Venzymatic activity; and cleaving the circular aptamer by theendonuclease V enzymatic activity to reduce or eliminate binding of thecircular oligonucleotide aptamer to the DNA polymerase, therebyactivating the DNA synthesis activity of the DNA polymerase, to increaseDNA synthesis in the reaction mixture. In the methods, cleaving may befacilitated using a reaction temperature that facilitates both DNApolymerase activity and the endonuclease V enzymatic activity. In themethods, cleaving may be facilitated by increasing the temperature ofthe reaction mixture from a first temperature to a second temperaturethat more strongly facilitates the endonuclease V enzymatic activity. Inthe methods, providing a reaction mixture suitable for DNA synthesis maycomprise dissolving a dried form of at least one of said (i) DNApolymerase, (ii) endonuclease V-cleavable circular oligonucleotideaptamer, and (iii) endonuclease V enzymatic activity into an aqueoussolution. DNA synthesis in the methods may result in DNA amplificationin the reaction mixture (e.g., wherein the DNA amplification comprisesPCR) DNA synthesis in the methods may comprise an isothermalamplification reaction. The methods may comprise detecting the presenceof a target DNA and/or measuring an amount of a target DNA in thereaction mixture. In the methods, the circular oligonucleotide aptamermay comprise one or more deoxyinosine nucleotides. In the methods, thecircular oligonucleotide aptamer may have a stem-loop structure, whereinone or more deoxyinosine nucleotides may be incorporated into the stemsegment of the stem-loop structure, and/or wherein one or moredeoxyinosine nucleotides may be incorporated into the loop segment ofthe stem-loop structure. In the methods, the loop of the stem-loopstructure, may, for example, comprise a nucleotide sequence selectedfrom the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23),5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:25). In the methods, the loop of the stem-loop structure may, forexample, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′(SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′(SEQ ID NO:21). In the methods, the endonuclease V enzymatic activitymay comprise, for example, Thermotoga maritima endonuclease V enzymaticactivity.

Additional aspects provide kits for activating an aptamer-inactivatedDNA polymerase, comprising: an endonuclease V enzymatic activity, and aDNA polymerase-binding circular oligonucleotide aptamer cleavable by anendonuclease V enzymatic activity. In the kits, the circularoligonucleotide aptamer may comprise one or more deoxyinosinenucleotides. In the kits, the circular oligonucleotide aptamer maycomprise a stem-loop structure. In the kits, one or more deoxyinosinenucleotides may be located in the stem segment of the stem-loopstructure, and/or one or more deoxyinosine nucleotides may be located inthe loop segment of the stem-loop structure. In the kits, the loop ofthe stem-loop structure, may, for example, comprise a nucleotidesequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ IDNO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ IDNO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQID NO:25). In the kits, the loop of the stem-loop structure may, forexample, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′(SEQ ID NO: 19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′(SEQ ID NO:21). In the kits, the endonuclease V enzymatic activity maycomprise, for example, Thermotoga maritima endonuclease V enzymaticactivity.

Further aspects provide reaction mixtures for use in a method of DNAsynthesis, which reaction mixture comprises: a DNA polymerase; anendonuclease V-cleavable circular oligonucleotide aptamer that bindsreversibly to the DNA polymerase, wherein the circular oligonucleotideaptamer is present in an amount effective to inhibit DNA synthesisactivity of the DNA polymerase in the reaction mixture, and anendonuclease V enzymatic activity capable of cleaving (or effective tocleave) the circular oligonucleotide aptamer to reduce or eliminatebinding of the circular oligonucleotide aptamer to the DNA polymerase,thereby activating the DNA synthesis activity of the DNA polymerase. Inthe reaction mixtures, the DNA polymerase activity and/or theendonuclease V enzymatic activity may be temperature-dependent. In thereaction mixtures, the endonuclease V enzymatic activity may increasewhen the reaction mixture is heated from a first temperature value to asecond temperature value that promotes the endonuclease V enzymaticactivity. In the reaction mixtures, the DNA polymerase, oligonucleotideaptamer, and endonuclease V enzymatic activity may be in a dried state.In the reaction mixtures, the circular oligonucleotide aptamer maycomprise one or more deoxyinosine nucleotides. In the reaction mixtures,the circular oligonucleotide aptamer may comprise a stem-loop structure.In the reaction mixtures, one or more deoxyinosine nucleotides may belocated in the stem segment of the stem-loop structure, and/or one ormore deoxyinosine nucleotides may be located in the loop segment of thestem-loop structure. In the reaction mixtures, the loop of the stem-loopstructure, may, for example, comprise a nucleotide sequence selectedfrom the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23),5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:25). In the reaction mixtures, the loop of the stem-loop structuremay, for example, comprise one of the nucleotide sequences5′-TTCITAGCGTTT-3′ (SEQ ID NO: 19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the reaction mixtures, theendonuclease V enzymatic activity may comprise, for example, Thermotogamaritima endonuclease V enzymatic activity. The reaction mixtures mayfurther comprise one or more of dATP, dCTP, dGTP, and/or dTTP, and/orMg²⁺ ion.

Yet further aspects provide circular oligonucleotide aptamers,comprising a nucleic acid sequence that forms a hairpin structure,having a stem and a loop portion, that binds to a DNA polymerase,wherein the stem and/or the loop portion comprises one or moredeoxyinosine nucleotides, and wherein the loop portion comprises anucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO: 18) that may besubstituted with deoxyinosine at one or more of positions 4, 5, and 7.In the oligonucleotide aptamers, the loop portion may comprise thenucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO: 18). In theoligonucleotide aptamers, one or more deoxyinosine nucleotides may belocated in the stem segment of the stem-loop structure, and/or one ormore deoxyinosine nucleotides may be located in the loop segment of thestem-loop structure. In the oligonucleotide aptamers, the loop of thestem-loop structure, may, for example, comprise a nucleotide sequenceselected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTT-3′ (SEQ ID NO:21),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23),5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:25). In the circular oligonucleotide aptamers, the loop of thestem-loop structure may, for example, comprise one of the nucleotidesequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ IDNO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the oligonucleotideaptamers, the one or more deoxyinosine nucleotides may render thecircular oligonucleotide aptamer cleavable by an endonuclease Venzymatic activity (e.g., wherein the endonuclease V enzymatic activitymay comprise Thermotoga maritima endonuclease V enzymatic activity).

Still further aspects provide compositions comprising a DNA polymerasecomplexed with a circular oligonuclcotide aptamer as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, according to particular exemplary aspects, a portion of ahuman β2-microglobulin gene sequence (SEQ ID NO:4), forward and reverseprimers (SEQ ID NOS: 1-2, respectively) and a 22-mer fluorescent probe(SEQ ID NO:3), which were used in exemplary 5′-nuclease PCR assays ofthe present invention from which exemplary results are shown in FIGS. 3and 5. The primers and probe are shown aligned with an amplifiedβ2-microglobulin fragment sequence in 5′-3′ orientation as indicated.

FIG. 2 shows, according to particular exemplary aspects, structures ofcircular stem-loop deoxvribonucleotide aptamers (shown at the rightcolumn of FIG. 2 under “Aptamer in circular form”) used in exemplary5′-nuclease PCR assays of the present invention from which exemplaryresults are shown in FIGS. 3A and 3B. The central column of FIG. 2 showslinear oligonucleotide sequences used to prepare the correspondingcircular aptamers SEQ ID NOS:6-11 as described herein in Example 2. Theaptamer SEQ ID NO:5 (shown at the top right of FIG. 2) is a noncircularanalog of aptamer SEQ ID NO:6 used in experiments to obtain the resultsshown in FIGS. 3A and 3B illustrating the effect of circularization onthe aptamer activity of deactivating DNA polymerase. The symbol “ρ”indicates 5′-phosphate moiety. The symbol “I” indicates the presence ofdeoxyriboinosine nucleotides in aptamers SEQ ID NOS:7-11 that were usedin the experiments with endonuclease V. Aptamers SEQ ID NOS:7 and 8incorporate deoxyinosine within the 5′-3′ duplex strand whereas aptamersSEQ ID NOS:9-11 incorporate deoxyinosine within the 3′-5′ duplex strand.Arrows point to the expected endonuclease V cleavage positions.

FIGS. 3A and 3B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual aptamers listed inFIG. 2. Sequences of the amplified β2-microglobulin template, primersand 22-mer FRET probe used in these PCR assays are as shown in FIG. 1.Dashed lines are real-time curves obtained in the absence of anyaptamer. Experiments were conducted in the absence (FIG. 3A) or presence(FIG. 3B) of T. maritima endonuclease V. Experimental details areprovided below in “Example 3.”

FIG. 4 shows, according to particular exemplary aspects, structures offour additional circular stem-loop deoxyribonucleotide aptamers SEQ IDNOS: 12-15 (right column of FIG. 4), which are derivatives of thecircular aptamer SEQ ID NO:6 of FIG. 2 and incorporate deoxyinosinenucleotide (“1”) in the loop portion at various indicated positions.These aptamers were used in the 5′-nuclease PCR assays from whichexemplary results are shown in FIGS. 5A and 5B. The central column ofFIG. 4 shows linear oligonucleotide sequences used to prepare thecorresponding circular aptamers SEQ ID NOS: 12-15 (shown at the rightcolumn of FIG. 4 under “Aptamer in circular form”), as described hereinin Example 2. The symbol “ρ” indicates a 5′-phosphate moiety. Arrowspoint to the expected endonuclease V cleavage positions.

FIGS. 5A and 5B show, according to particular exemplary aspects, theresults of fluorescence monitoring of reaction mixtures during PCR(real-time curves) in the presence of the individual circular aptamerslisted in FIG. 4. Sequences of the amplified β2-microglobulin template,primers and 22-mer FRET probe used in the assays are as shown in FIG. 1.Dashed lines are real-time curves obtained in the absence of anyaptamer. Experiments were conducted in the absence (FIG. 5A) or presence(FIG. 5B) of T. maritima endonuclease V. Experimental details areprovided herein below under “Example 3.”

FIG. 6 shows, according to particular exemplary aspects, a reactionscheme used in the assays of FIGS. 7A through 7F to detect and measureDNA polymerase activity. The depicted hairpin-like fluorescent probe SEDID NO:16 was designed to have a G/C-rich stem in the duplex segment anda 5′- . . . GAA . . . hairpin-stabilizing loop to provide for use atelevated temperatures (e.g., up to 60-65° C.) (e.g., see Yoshizawa S. etal, 1994). Extension of this hairpin-like probe in a reaction buffer inthe presence of deoxyribonucleoside 5′-triphosphates (dNTPs) and a DNApolymerase results in a fluorescent signal that directly correlates withthe polymerase activity in the reaction.

FIGS. 7A through 7F show, according to particular exemplary aspects,endonuclease V-induced activation of Taq (FIG. 7D), Phusion® (FIG. 7B),Q5® (FIG. 7C), Vent® (FIG. 7A), Deep Vent® (FIG. 7E) and Bst largefragment (FIG. 7F) DNA polymerases that were initially deactivated(i.e., “inhibited” or “blocked”) by the presence of aptamer SEQ ID NO:12 (♦ curves). FIGS. 7A through 7F also show the change of fluorescencewith time in the absence of endonuclease V for the aptamer-blocked (∘curves) and unblocked (□ curves) DNA polymerase. The DNA polymeraseactivity was monitored by extension of the self-priming hairpin-likefluorescent probe SEQ ID NO: 16 (see FIG. 6), which was present in thereaction mixture with all four dNTPs in a magnesium-containing buffer.In all cases experiments were performed at 60 or 65° C., as indicated ineach Figure. The structure of aptamer SEQ ID NO: 12 is shown in FIG. 4,and details of the experimental setup, results analysis and conclusionsare provided below in “Example 4.”

DETAILED DESCRIPTION Definitions

Terms and symbols of biochemistry, nucleic acid chemistry, molecularbiology and molecular genetics used herein follow those of standardtreatises and texts in the field (e.g., Sambrook, J., et al, 1989;Komberg, A. and Baker, T., 1992; Gait, M. J., ed., 1984; Lehninger, A.L., 1975; Eckstein F., ed., 1991, and the like). To facilitateunderstanding of particular exemplary aspects of the invention, a numberof terms are discussed below.

In particular aspects, “aptamer” or “oligonucleotide aptamer” refersherein to an oligonucleotide that is capable of binding to a DNApolymerase and blocking its DNA synthesis enzymatic activity. Theaptamers can be linear circular molecules and/or capable to formsecondary structures such as hairpin or stem-loop structures, etc.Examples of aptamers and methods of selection (design) can be found, forinstance, in Yakimovich, O. Yu., et al. (2003); Jayasena, S. D. (1999);U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D, which areincorporated here by reference. As used herein, the term “circular”refers to oligonucleotide that forms a closed loop and has no ends. Thephrase “aptamer, that binds to the DNA polymerase, in an amountsufficient to inhibit DNA synthesis activity of the DNA polymerase,” asused herein, means that the DNA synthesis activity of the DNA polymeraseis at least partially inhibited (e.g., inhibited to a level in the rangeof from about 1% to about 99.99%). Any level of aptamer inhibition ofthe DNA synthesis activity of the DNA polymerase can provide anadvantage for DNA synthesis, and thus according to particular preferredhot start aspects of the present invention, the DNA synthesis activityof the DNA polymerase is substantially inhibited (e.g., inhibited to alevel in the range of about 80% to 99.99%, or to any subrange or leveltherein), or completely (100%) inhibited, providing an advantage overother ‘hot start’ technologies (e.g., Paul N., et al., 2010). Likewise,in the disclosed methods. “cleaving the aptamer by the Endonuclease Venzymatic activity to reduce or eliminate the binding of theoligonucleotide aptamer to the DNA polymerase and activate the DNAsynthesis activity of the DNA polymerase” is preferably complete (100%)or substantially complete (e.g., inhibited to a level in the range ofabout 80% to 99.99%, or to any subrange or level therein), but can bepartial (e.g., inhibited to a level in the range of from about 1% toabout 99.99%), as exemplified herein (e.g., FIGS. 3, 5, 7, and 9).

An oligonucleotide aptamer may comprise ribo- or 2′-deoxyribonucleotidesor a combination thereof. Oligonucleotide aptamers may be modified.Regarding the aptamers herein, the terms “modified” and “modification”are used in two different aspects, wherein the aptamers can be (i)modified synthetically, e.g. during the oligonucleotide synthesis, and(ii) enzymatically-modified in the context of or during DNA synthesisreactions. Synthetically, the aptamers may incorporate any kind and/ornumber of structural modifications across the length of the aptamer(e.g., in the middle or at the ends of the oligonucleotide chain). Theterm “structural modifications” refers to any chemical substances suchas atoms, moieties, residues, polymers, linkers or nucleotide analogs,etc., which are usually of a synthetic nature and which are not commonlypresent in naturally-occurring nucleic acids. As used herein, the term“structural modifications” also include nucleoside or nucleotide analogswhich are rarely present in naturally-occurring nucleic acids includingbut not limited to inosinc, 5-bromouracil, 5-methylcytosine,5-iodouracil, 2-aminoadenosine, 6-methyladenosine, pseudouridine,deoxyuridine, and the like. In particular embodiments, the aptamersincorporate one or more deoxyinosine (deoxyriboinosine) nucleotides thatenable an endonuclease V enzyme to recognize the oligonucleotide aptameras a substrate and modify its structure by cleavage. Nucleotides in theaptamers may be modified at the phosphates, sugar moieties or nucleotidebases. The structural modifications can be “duplex-stabilizingmodifications.” “Duplex-stabilizing modifications” refer to structuralmodifications, the presence of which in double-stranded nucleic acidsprovides a duplex-stabilizing effect when compared in thermal stability,usually measured as “Tm,” with respective nucleic acid complexes thathave no such structural modification and, e.g., comprise naturalnucleotides. Duplex-stabilizing modifications are structuralmodifications that are most commonly applied in synthesis of probes andprimers, as represented by modified nucleotides and ‘tails’ likeintercalators and minor groove binders as, for example, disclosed inU.S. Pat. No. 8,349,556 to Kutvavin, I. V.; U.S. Pat. No. 7,794,945 toHedgpeth, J., et al.; U.S. Pat. No. 6,127,121 to Meyer, Jr., R. B., etal.; U.S. Pat. No. 5,801,155 to Kutyavin, I. V., et al, and thereferences cited in. Duplex-stabilizing modifications can be used toprepare aptamers of the invention, for example, to improve thermalstability of stem (duplex) structures of hairpin-like aptamers. Inpreferred methods of the invention, the oligonucleotide aptamers aremodified in (e.g., during) DNA synthesis reactions using enzymaticactivity of one or more aptamer-modifying enzyme(s). In this aspect, theterms “modify.” “modification,” and “structural modifications” meanchanges in the initial chemical structure of the aptamers. The change istriggered by Endonuclease V enzymes, resulting in a cleavage of one ormore phosphodiester bonds. In the methods of the invention, theseEndonuclease V-triggered structural modifications reduce or eliminatethe ability of the oligonucleotide aptamer to bind to the DNA polymeraseand block or reduce its activity in the reaction. In certain aspects,oligonucleotide aptamers comprise one or more deoxyinosine nucleotides,and aptamer modification results from aptamer cleavage (phosphodiesterbond cleavage) by endonuclease V, at the second phosphodiester bond inthe DNA strand on the 3′ side of a deoxyinosine nucleotide.

In particular aspects, the term “secondary structure” refers to anintramolecular complex formation of one sequence in a poly- oroligonucleotide with another sequence in the same polymer due tocomplete or partial complementarity between these two sequences formedbased on the principal rules of Watson-Crick base pairing. The terms“hairpin” structure and “stem-loop” structure as referred to hereindescribe elements of secondary structure, and both terms refer to adouble-helical region (stem) formed by base pairing betweencomplementary sequences within a single strand RNA or DNA.

As used herein, the term “nuclease” refers to an enzyme that expresses aphosphomonoesterase or phosphodiesterase activity and is capable ofcleaving a phosphoester bond in compounds such as R′—O—P(O)OH)₂ andR′—O—P(O)(OH)—O—R,″ resulting in products R′—OH+P(O)(OH)₃ andR′—OH+P(O)(OH)₂—O—R″ (or R″—OH+P(O)(OH)₂—O—R′), respectively, andwherein R′ and R″ may be moieties of any structure which are notnecessarily of a nucleotide nature. The term “nucleases” incorporatesboth “exo” and “endo” nucleases. In certain aspects, endonuclease V isused to cleave phosphodiester bonds of oligonucleotide aptamerscomprising one or more deoxyinosine nucleotides.

The term “aptamer-modifying enzymatic activity” refers to anaptamer-modifying enzyme or mixture of aptamer-modifying enzymes whichrecognize an aptamer of the invention as a substrate and modifies itsstructure so that the inhibitory activity of the aptamer issubstantially disabled. In particular aspects, this recognition isdirected by the presence of one or more deoxyinosine nucleotides withinthe aptamer structure. These aptamer structural modifications caused byendonuclease V enzymatic activity reduce the ability of theoligonucleotide aptamer to bind to the DNA polymerase and to block orreduce the polymerase activity in the reaction mixture.

As used herein, the term “deoxyinosine” refers to deoxyinosine andstructural modifications of deoxyinosine, including but not limited to7-deaza-deoxysine, 8-aza-7-deaza-deoxyinosine and other structuralmodifications of deoxyinosine, and including various ribonucleotidederivatives thereof, which can be recognized and cleaved by anendonuclease V enzymatic activity.

As used herein, the term “deoxyuridine” refers to deoxyuridine andstructural modifications of deoxyuridine, including but not limited tovarious ribonucleotide derivatives thereof, that can be recognized andcleaved by an endonuclease V enzymatic activity.

As used herein, the term “endonuclease V” refers to repair enzymes thatrecognize deoxyinosine in DNA, and hydrolyze the second phosphodiesterbond in the DNA strand on the 3′ side of a deoxyinosine nucleotide (see,e.g., U.S. Pat. No. 8,143,006 to Kutyavin, I. V.).

The term “DNA polymerase” refers to an enzyme that catalyzes synthesisof deoxyribo nucleic acids (DNAs), most commonly double-stranded DNAs,using single-stranded DNAs as “templates.” The DNA synthesis is usuallyinitiated by an oligonucleotide primer that is hybridized to acomplementary template strand. Starting from this template-hybridizedprimer, DNA polymerase creates a Watson-Crick complementary strand inthe presence of 2′-deoxyribonucleotide 5′-triphosphates (dNTPs). Theterm “DNA polymerase,” as used herein, also incorporates “reversetranscriptases,” enzymes which can perform DNA synthesis usingsingle-stranded ribonucleic acids (RNAs) as template strands.

“Polynucleotide” and “oligonucleotide” are used herein interchangeablyand in each case means a linear polymer of nucleotide monomers.Polynucleotides typically range in size from a few monomeric units,e.g., 5-60, when they are usually referred to as “oligonucleotides,” toseveral thousand monomeric units. The exact size will depend on manyfactors, which in turn depends on the ultimate function or use of theoligonucleotide. The oligonucleotides may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription, ora combination thereof. Unless otherwise specified, whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters, for example, “TTCTTAGCGTTT (SEQ ID NO:18),” it is understoodherein, unless otherwise specified in the text, that the nucleotides arein 5′→3′ order from left to right and that “A” denotes deoxyadenosine,“C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotesdeoxythymidine. Usually DNA polynucleotides comprise these fourdeoxyribonucleosides linked by phosphodiester linkage whereas RNAcomprises uridine (“U”) in place of ‘T’ for the ribose counterparts.

The terms “oligonucleotide primer” and/or “primer” refer to asingle-stranded DNA or RNA molecule that hybridizes to a complementarytarget nucleic acid and serves to prime enzymatic synthesis of a secondnucleic acid strand in the presence of a DNA polymerase. In this case,the target nucleic acid “serves as a template” for the oligonucleotideprimer.

In particular aspects, the terms “complementary” or “complementarity”are used herein in reference to the polynucleotide base-pairing rules.Double-stranded DNA, for example, consists of base pairs wherein, forexample, G complexes or pairs with C via formation of a three hydrogenbond complex, and A complexes or pairs with T via formation of a twohydrogen bond complex, such that G is regarded as being complementary toC, and A is regarded as being complementary to T. In this sense, forexample, an oligonucleotide 5′-GATTTC-3′ is complementary to thesequence 3′-CTAAAG-5′ via intrastrand G:C and A:T hydrogen bondinginteractions. Complementarity may be “partial” or “complete.” In partialcomplementarity, only some of the nucleic acids bases are matchedaccording to the base pairing rules. “Complementarity” may also be usedin reference to individual nucleotides and oligonucleotide sequenceswithin the context of polynucleotides (e.g., inter-strandcomplementarity). The terms “complementary” and “complementarity” referto the most common type of complementarity in nucleic acids, namely,Watson-Crick base pairing as described above, although theoligonucleotides may alternately participate in other types of“non-canonical” pairings like Hoogsteen, wobble and G-T mismatchpairing.

The term “natural nucleosides” refers to the four standard2′-deoxyribonucleosides (usually named herein as “deoxynucleosides” or“deoxyribonucleosides”) that are found in DNAs isolated from naturalsources. Natural nucleosides are deoxyadenosine, deoxycytidine,deoxyguanosine, and deoxythymidine. The term also encompasses theirribose counterparts, with uridine (U) in place of thymidine. The samename variations are applied herein to “natural nucleotides.”

As used herein, the terms “unnatural nucleosides” or “modifiednucleosides” refer to nucleoside analogs that are different in theirstructure from those natural nucleosides for DNA and RNA polymers. Somenaturally occurring nucleic acids contain nucleosides that arestructurally different from the natural nucleosides defined above, forexample, DNAs of eukaryotes may incorporate 5-methyl-cytosine, and tRNAscontain many nucleoside analogs. However, as used herein, the terms“unnatural nucleosides” or “modified nucleosides” encompasses thesenucleoside modifications even though they can be found in naturalsources.

The term “reaction mixture” generally means herein a solution containingall necessary reactants for performing DNA synthesis such as a DNApolymerase, oligonucleotide primer(s), template polynucleotide,deoxyribonucleoside 5′-triphosphates, reaction cofactors (e.g.,magnesium or manganese ions), etc. The reaction mixture can incorporateother reaction components that help to improve DNA synthesis (e.g.,buffering and salt components, detergents, proteins like bovine serumalbumin (BSA), scavengers, etc.) or components that are necessary fordetection of the newly synthesized DNA molecules such as, for example,fluorescent dyes and oligonucleotide probes. A reaction mixture isusually prepared at low temperatures at which enzymatic components areinactive, for example, by mixing the components on ice at ˜0° C. Whenthe reactions are ready, the mixtures can be heated to the desiredreaction temperatures. In this aspect, the term “reaction temperature”refers to a temperature or a temperature range at which DNA synthesis isperformed. In case of PCR reactions, it is usually taken as the lowestthermo-cycling temperature, commonly called the annealing temperature.

“dNTPs” is an abbreviation of a mixture of two or more of the fournatural deoxynucleoside 5′-triphosphates that are useful to facilitateprimer extension with a DNA polymerase and/or amplification.Respectively, the abbreviations “dATP,” “dCTP,” “dGTP,” and “dTTP”correspond to the individual nucleotides. In some embodiments, the fourdNTPs are present at equal concentrations. In other embodiments, theconcentrations of the dNTPs are not all identical. In some embodiments,fewer than all four dNTPs are present. For example, only one dNTP may bepresent, or a pair-wise combinations, or three of four dNTPs may bepresent in the mixture.

In some aspects, “amplification” and “amplifying” deoxyribonucleicacids, in general, refer to a procedure wherein multiple copies of DNAof interest are generated. The DNA amplification can be performed at aconstant temperature using “isothermal amplification reactions.”Examples of isothermal amplification reactions include, but are notlimited to, Strand Displacement Amplification (SDA) (U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta. N., et al.), Rolling Circle amplification (RCA) (U.S. Pat.No. 5,854,033 to Lizardi. P.), Loop-Mediated Amplification (LMA) (U.S.Pat. No. 6,410,278 to Notomi, T. and Hase. T.), isothermal amplificationusing chimeric or composite RNA DNA primers (U.S. Pat. No. 5,824,517 toCleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kum, N.),Nucleic Acid Sequence-Based Amplification (NASBA) (U.S. Pat. No.6,063,603 to Davey, C. and Malek, L. T.), and many other methods.

“PCR” is an abbreviation of “polymerase chain reaction,” anart-recognized nucleic acid amplification technology (e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Commonly used PCRprotocol employs two oligonucleotide primers, one for each strand,designed such that extension of one primer provides a template for theother primer in the next PCR cycle. Generally, a PCR reaction consistsof repetitions (cycles) of (i) a denaturation step that separates thestrands of a double-stranded nucleic acid, followed by (ii) an annealingstep, which allows primers to anneal to positions flanking a sequence ofinterest on a separated strand, and then (iii) an extension step thatextends the primers in a 5′ to 3′ direction, thereby forming a nucleicacid fragment complementary to the target sequence. Each of the abovesteps may be conducted at a different temperature using an automatedthermocycler. The PCR cycles can be repeated as often as desiredresulting in an exponential accumulation of a target DNA fragment whosetermini are usually defined by the 5′-ends of the primers used. Althoughconditions of PCR can vary in a broad range, a double-stranded targetnucleic acid is usually denatured at a temperature of >90° C., primersare annealed at a temperature in the range of about 50-75° C., and theextension is preferably performed in a 72-75° C. temperature range. InPCR methods, the annealing and extension can be combined into one stage(i.e., using a single temperature). The term “PCR” encompasses itsnumerous derivatives such as “RT-PCR,” “real-time PCR,” “nested PCR,”“quantitative PCR,” “multiplexed PCR,” “asymmetric PCR,” and the like.

“Real-time detection” means an amplification reaction for which theamount of reaction product, i.e., target nucleic acid, is monitored asthe reaction proceeds. Real-time detection is possible when alldetection components are available during the amplification and thereaction composition and conditions support both stages of the reaction,the amplification and the detection.

As used herein, the term “kit” refers to any system for deliveringmaterials. In the context of reaction assays, such delivery systemsinclude elements allowing the storage, transport, or delivery ofreaction components such as oligonucleotides, buffering components,additives, reaction enhancers, enzymes, and the like in the appropriatecontainers from one location to another commonly provided with writteninstructions for performing the assay. Kits may include one or moreenclosures or boxes containing the relevant reaction reagents andsupporting materials. The kit may comprise two or more separatecontainers wherein each of those containers includes a portion of thetotal kit components. The containers may be delivered to the intendedrecipient together or separately.

In general, the term “design” in the context of the methods has broadmeaning and in certain respects is equivalent to the term “selection.”For example, the terms “primer design” and “aptamer design” can mean orencompass selection of a particular oligonucleotide structure includingthe nucleotide primary sequence and structural modifications (e.g.,labels, modified nucleotides, linkers, etc.). In particular aspects, theterms “system design” and “assay design” relate to the selection of any,sometimes not necessarily to a particular, methods including allreaction conditions (e.g., temperature, salt, pH, enzymes, including theaptamer-modifying enzymes and DNA polymerase, oligonucleotide componentconcentrations, etc.), structural parameters (e.g., length and positionof primers and probes, design of specialty sequences, etc.) and assayderivative forms (e.g., post-amplification, real time, immobilized, FRETdetection schemes, etc.) chosen to amplify and/or to detect the nucleicacids of interest.

Reversible Blocking of DNA Synthesis Activity of DNA Polymerases UsingCircular Oligonucleotide Aptamers.

Prior use of oligonucleotide aptamers during DNA synthesis haveattempted to block DNA polymerase activity, preferably completely, atlow temperatures, while releasing (activating) the DNA polymeraseactivity, preferably completely, at an elevated reaction temperature. Itis difficult, however, to achieve complete ‘block-and-release’ formatsusing conventional aptamer-based methods (e.g., Yakimovich, O. Yu., etal. (2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L.and Jayasena, S. D. (1997)). Effective blockage of DNA polymerase at lowtemperatures commonly leads to ineffective release of the enzyme at theelevated reaction temperature and vice versa. Aspects of the presentinvention provide a solution to this long-standing problem in the art.As in the conventional approaches cited above, the DNA polymeraseactivity is blocked or reduced in methods of the invention by thepresence of a circular oligonucleotide aptamer that binds to the DNApolymerase, blocking the DNA synthesis activity of the DNA polymerase.Unlike prior art techniques, however, in methods of the invention, theaptamer-inactivated DNA polymerase is activated by providing to a DNAsynthesis reaction mixture an endonuclease V enzyme activity thatrecognizes the circular oligonucleotide aptamer as a substrate andcleaves its structure. This cleavage reduces or eliminates the bindingof the circular oligonucleotide aptamer to the DNA polymerase andthereby reactivates the DNA synthesis activity of the DNA polymerase.

In some embodiments of the invention, activation of anaptamer-inactivated DNA polymerase in a reaction mixture, comprising (i)a DNA polymerase, (ii) circular oligonucleotide aptamer in an amounteffective to inhibit the DNA synthesis activity of the DNA polymerase,(iii) an endonuclease V aptamer-modifying enzyme and other componentsnecessary for DNA synthesis, is facilitated using a reaction temperaturethat accelerates (or facilitates) both DNA polymerase and endonuclease Venzymatic activities. For example, the reaction mixture can be preparedat low temperature (first temperature) at which a DNA polymerase iseffectively blocked by an aptamer and an aptamer-modifying enzyme,endonuclease V, has sufficiently reduced or preferably no activity(e.g., at 0° C.), and then the activation of the aptamer-inactivated DNApolymerase is facilitated by heating the reaction to a temperature(second temperature) that accelerates or facilitates theaptamer-modifying endonuclease V enzymatic activity. If necessary, a DNApolymerase can be activated by the endonuclease V enzyme(s) at anytemperature below the reaction temperature for DNA synthesis. This canbe applied, for example, when a particular endonuclease V enzyme isunstable at the reaction temperature for DNA synthesis, for example, dueto denaturation. In this case, the DNA polymerase is first activated atan intermediate temperature wherein the endonuclease V is active andthen heated to the reaction temperature to perform DNA synthesis.

In some aspects, the reaction mixture is created by addition of aqueoussolution to one or more reaction components which are initially in adried state as disclosed, for example, in U.S. Pat. No. 3,721,725 toBriggs, A. R. and Maxwell, T. J. (1973) (incorporated herein byreference). For example, in some methods of the invention for DNAamplification and detection, the aqueous solution can be a samplesolution or solution that contains one or more polynucleotide templatesfor DNA synthesis whereas all other reaction components such as the DNApolymerase, aptamer, endonuclease V enzyme, dNTPs, catalytic cofactorssuch as magnesium (Mg2+) and/or manganese (Mn2+) ion (e.g., provided asa chloride salt), buffering components, detergents, proteins like bovineserum albumin (BSA), scavengers, etc., are initially provided in a drystate.

In some aspects, DNA synthesis results in DNA amplification in thereaction mixture. The DNA amplification can be an isothermalamplification reaction, for example, as described in U.S. Pat. No.5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 toDattagupta, N., et al.; U.S. Pat. No. 5,854,033 to Lizardi, P.; U.S.Pat. No. 6,410,278 to Notomi, T. and Hase, T.; U.S. Pat. No. 5,824,517to Cleuziat, P. and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.;U.S. Pat. No. 6,063,603 to Davey, C. and Malek, L. T., and many othermethods. In other aspects, the DNA amplification can be a PCR reaction(e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Inmethods of the invention, the DNA amplification is performed fordetection as well as measuring an amount of a target DNA in the reactionmixture.

As has been established in the art (e.g., Yao, M., Kow, Y. W., 1997), anumber of nucleotide modifications can be recognized by the endonucleaseV enzymes to provide for the oligonucleotide cleavage. For example, anoligonucleotide aptamer that contains one or more deoxyuridinenucleotide(s) as described, for example, in Yao, M., Kow, Y. W., 1997,can be used in methods, compositions and kits of the present invention,such that contact of such aptamers with an endonuclease V enzymaticactivity causes cleavage of the deoxyuridine-containing aptamer.However, the preferred modified nucleotide in aptamers is one or moredeoxyinosine nucleotide(s). The endonuclease V enzymes of the presentinvention can cleave single-stranded and double-strandedoligonucleotides. Therefore the circular aptamers of the presentinvention can be both, single and double-stranded. In particularembodiments, the circular aptamers of the invention are stem-loop orhairpin-like molecules. Effective use of these two exemplarycombinations (endonuclease V)+(deoxyinosine-incorporating circularaptamers) is illustrated in the Examples provided herein using, inparticular, hairpin-like structures (FIGS. 2-6; as described in workingExample 3 herein below). Deoxyinosine nucleotide can be located eitherin loop or stem fragments of the hairpin-like circular aptamers of theinvention. Preferred locations of deoxyinosine modifications in circularaptamers of the present invention are described herein.

The oligonucleotide aptamers as well as oligonucleotide primers andprobes can be prepared by any method of oligonucleotide synthesisselected by a person of ordinary skill in the art, such as methods thatutilize (2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites (Example 1).Protected nucleotides and derivatives, linkers, dyes, tails, solidsupports, and other appropriate components can be prepared by methods oforganic chemistry or obtained from market providers such as, forexample, Glen Research and Biosearch Technologies. Suppliers such asIntegrated DNA Technologies and Biosearch Technologies also offeroligonucleotide custom synthesis including numerous structuralmodifications such as deoxyinosine and deoxyuridine. In the methods ofthe invention, selection of an aptamer structure, including one or moredeoxyinosine nucleotides, is intended to achieve (i) completedeactivation of the DNA polymerase at the initial reaction assemblytemperature, (ii) complete or substantially complete (or as much aspossible) deactivation of the DNA polymerase at the elevated reactiontemperature for DNA synthesis, and (iii) substantially complete orcomplete (or as much as possible) reactivation of this enzyme at the DNAsynthesis reaction temperature once the circular aptamer has beenmodified by the endonuclease V enzymatic activity. Generally,endonuclease V enzymes do not interfere with DNA synthesis (primerextension) or DNA amplification. The location and number of deoxyinosinenucleotides within a circular aptamer, as well as the rate andefficiency of the endonuclease V enzymes is preferably taken intoconsideration. Preference is given to deoxyinosine locations within acircular aptamer that have little or no negative effect on stability ofthe aptamer-DNA polymerase complex, but sufficiently disturb thestructural integrity of the circular aptamer so that the cleaved aptamerdoes not bind to or inhibit the DNA polymerase. Results from circularaptamers containing deoxyinosine nucleotides (SEQ ID NOS:7-15 and 12-20)as the endonuclease V-recognition motif are illustrated in FIGS. 3, 5,and 7. Surprisingly, even a single base modification at numerouslocations showed excellent results in the exemplary assays.

According to the prior art (Yakimovich, O. Yu., et al. (2003). Jayasena,S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.),efficiency of hairpin-type aptamer binding to DNA polymerase isdetermined by (i) a loop segment, which is a substantially conservativesequence, and (ii) length of the stem duplex, which preferably needs tobe ˜19-20 base pairs or longer. In addition, the present inventors havefound that in addition to the sequence, the circularization of thehairpin-like aptamers can be another important factor affecting thestability of an aptamer-polymerase complex. For example, the stemsequence of aptamer SEQ ID NO:5 (FIG. 2) in the present working Examplesmostly comprises A and T nucleotides. As illustrated in FIGS. 3A and 3B,this aptamer SEQ ID NO:5 has little, if any DNA-polymerase deactivatingaffect during PCR, whereas it circular analog SEQ ID NO:6 was veryeffective in DNA-polymerase deactivation (FIGS. 3 and 5). Moreover, thedeoxyinosine-incorporating circular analog SEQ ID NO: 12 was veryeffective in blocking not only Taq (FIGS. 5A and 5B), but also manyother DNA polymerases (FIG. 7). Out of six DNA polymerases investigated,only Bst polymerase (large fragment) was not inactivated by the circularaptamer SEQ ID NO:12. Analysis of FIG. 7 points to additional surprisingresults. First, regardless of the difference in reaction temperature,circular aptamer SEQ ID NO:12 blocked Phusion® polymerase much moreefficiently at 65° C. than Taq polymerase at 60° C. According toadditional surprising aspects of the invention, therefore, thepolymerase-blocking efficiency of the circular aptamer SEQ ID NO: 12varies for the DNA polymerases investigated. Second, the duplex sequencein an aptamer of the invention can be further optimized by base pairchanges for even better polymerase blockage in each particular case.Third, using the sequence of circular aptamer SEQ ID NO:6 as an origin,sequence optimization for strongest binding can be performed for everyDNA polymerase known in the art, although for some DNA polymerases likeBst, optimization may require alterations in the loop segment of theaptamer as well as in the stem. In this sense, the present disclosurealso provides methods of screening for more optimal aptamers for use inthe disclosed methods.

Circular aptamers of the invention, whether single-stranded ordouble-stranded, can contain any number of modified nucleotides,internal linkers and moieties, and other structural modifications aslong as these modifications do not interfere with the DNA polymerasedeactivation and subsequent reactivation processes during DNA synthesis.Preference should be given to structural modifications that help todeactivate the DNA polymerase and do not adversely affect theendonuclease V enzyme activation reaction. Both loop and stem fragmentscan be modified in the circular hairpin-type aptamers. Although the loopsegments described in Yakimovich, O. Yu., et al. (2003), Jayasena, S. D.(1999), and U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena. S. D.,contain conserved sequence motifs, the results in FIG. 5 (Example 3)herein show that deoxyinosine substitution(s) at certain loop positionshave very little (SEQ ID NO:14) or no effect (SEQ ID NO:12) on aptamerperformance (FIG. 5). The circular hairpin-like aptamers used in theworking Examples of the present invention (structures shown in FIGS. 2and 4) were designed to incorporate one short 5′-GAA-3′ loop (secondloop) on the end of the double-stranded stem opposite the end having theconservative 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18) loop sequence. In methodsof the invention, this second loop can vary in length and nucleotidesequence and composition. It can be, for example, the same conservativesequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18), or a different sequenceand/or different length, etc.

Methods of the invention can be performed using one or more reactiontemperatures wherein a DNA polymerase and endonuclease V expresssuitable activity. Specificity of DNA synthesis is usually increased athigher temperatures, and therefore thermostable enzymes are usuallypreferred. The upper level of the reaction temperature can be selectedbased on the DNA polymerase stability. In cases when an endonuclease Vis not stable at a desired reaction temperature, the DNA polymeraseactivation can be initiated at a lower intermediate temperature whereinthe endonuclease V is stable and active and then raised to the desiredreaction temperature for DNA synthesis (primer extension). In someembodiments, a DNA polymerase is preferably first deactivated bycontacting (e.g., by combining or mixing) with an aptamer before otherreaction components of the DNA synthesis are added. Molar reactionconcentration of an aptamer applied should be at least equal to theconcentration of a DNA polymerase or preferably greater. Marketproviders commonly do not disclose the molar amount of the enzymes,therefore the precise excess of the aptamers over the DNA polymerasesused in Examples provided herein was not known. However, the aptamersused in the Examples below were estimated to be present in a range of˜40-80 fold, or greater, molar excess relative to DNA polymerase. Insome embodiments, the aptamer is present in a molar excess (ratio) overthe DNA polymerases of at least ˜5-fold, although the ratio can behigher or lower than 5-fold. The amounts of the enzymes, circularaptamers and other reaction components used in the reaction may beoptimized and depend on many factors including, but not limited toselection of the particular enzymes, enzymatic activities at thereaction temperature, reaction temperature itself, nature of thecircular aptamers, and their special endonuclease V recognition motifs(e.g., deoxyinosine or deoxyuridine, etc.) to provide for cleavage ofthe aptamers. Methods of the present invention can be particularlyuseful for so-called ‘fast’ PCR with a cycle time shorter than 20seconds.

In certain embodiments, methods of the invention can be practiced usinga kit comprising a DNA polymerase-binding circular oligonucleotideaptamer recognizable and modifiable by an endonuclease V enzymaticactivity, and an endonuclease V enzyme activity to provide for specificcleavage of the circular aptamer. The kit can also include a DNApolymerase that is initially deactivated by the circular oligonucleotideaptamer. Alternatively, the kit can include, in addition to theendonuclease V enzyme activity, a complex of the DNA polymerase with thecircular oligonucleotide aptamer, wherein the components of this complexare present at a specific and optimal molar ratio. As a matter ofconvenience, such a kit can include components allowing the storage,transport and other reaction components such as oligonucleotides,buffering components, additives, reaction enhancers, etc. The circularaptamers of the kits can be single-stranded or have a stem-loopstructure, and they can incorporate one or more special structuralfeatures. The kits can be used for the DNA synthesis, amplification aswell as the detection of the amplified DNA fragments.

In some embodiments, the invention includes a reaction mixturecomprising a DNA polymerase, a circular oligonucleotide aptamer thatbinds to the DNA polymerase and present in an amount effective toinhibit DNA synthesis activity of the DNA polymerase, an endonuclease Venzyme activity that is capable of cleaving the oligonucleotide aptamerto reduce or eliminate binding of the oligonucleotide aptamer to the DNApolymerase, and other reaction components necessary for DNA synthesis isalso a subject of the present invention. In some embodiments, thereaction mixture can be assembled using concentrated stock solutions ofone or more components, usually in water to provide the desiredcomponent concentration in the final mixture. Mixing is recommended tobe performed at a low temperature (e.g., close to 0° C.) at which theenzymes, particularly endonuclease V enzymes, are inactive. Preferably,the reaction mixture should be used for DNA synthesis soon afterpreparation. Storage of a fully assembled reaction mixture is notrecommended. However, reaction components including enzymes can retainactivity for a long time (days, months or even years) in a dried state.For example, in some embodiments of the invention, one or more of thecomponents for forming a mixture of the invention can be provided in adried form, such as dried beads as described, for example, in U.S. Pat.No. 3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973), including (butnot limited to) DNA polymerase, circular oligonucleotide aptamer, andendonuclease V enzyme activity such that one or more of the componentsis prepared in a form of dried beads as described, for example, in U.S.Pat. No. 3,721,725 to Briggs, A. R. and Maxwell, T. J. (1973). In someembodiments, the mixture comprises DNA polymerase, a circularoligonucleotide aptamer that binds to the DNA polymerase and present inan amount effective to inhibit DNA synthesis activity of the DNApolymerase, and an endonuclease V enzyme activity that is capable ofcleaving the oligonucleotide aptamer to reduce or eliminate binding ofthe circular oligonucleotide aptamer to the DNA polymerase, which aremixed together in a dried form.

Example 1 Synthesis of 5′-Phosphate-Incorporating Oligonucleotides.Noncircular Aptamer, Primers and Fluorescent Probes

Standard phosphoramidites, including modified nucleotide analogs such asdeoxyinosine phosphoramidite (Catalog Number: 10-1040-xx), aphosphoramidite for incorporation of 5′-phosphate moiety, solid supportsand reagents to perform solid support oligonucleotide synthesis, werepurchased from Glen Research. A 0.25 M 5-ethylthio-1H-tetrazole solutionwas used as a coupling agent. Oligonucleotides were synthesized eitheron AB1394 DNA synthesizer (Applied Biosystems) or MerMaid 6 DNAsynthesizer (BioAutomation Corporation) using protocols recommended bythe manufacturers for 0.2 μmole synthesis scales. Fluorescein (FAM)conjugated to 5-position of deoxyribouridine (U) of probe SEQ ID NO:29(FIG. 8) was introduced to the hairpin during oligonucleotide synthesisusing 5′-dimethoxytrityloxy-5-[N-((3′,6′-dipivaloylfluoresceinyl)-aminohexyl)-3-acrylimido]-2′-deoxyribouridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research, Catalog Number: 10-1056-xx). A 6-fluorescein reportingdye was incorporated onto the 5′-end, and a BHQ1 quencher was introducedto the 3′-end of probe SEQ ID NO:3 (FIG. 1) using respectivephosphoramidite and CPG from Biosearch Technologies (Catalog numbers:BNS-5025 and i-5041G). After the automated synthesis, oligonucleotideswere deprotected in aqueous 30% ammonia solution by incubation for 12hours at 55° C. or 2 hours at 70° C.

Tri-ON oligonucleotides were purified by HPLC on a reverse phase C18column (LUNA 5 μm, 100 A, 250×4.6 mm, Phenomenex Inc.) using gradient ofacetonitrile in 0.1 M triethyl ammonium acetate (pH 8.0) or carbonate(pH 8.5) buffer with flow rate of 1 ml/min. A gradient profile includingwashing stage 0→14% (10 see), 14→45% (23 min), 45→90% (10 min), 90→90%(5 min), 90→0% (30 sec), 0→0% (7 min) was applied for purification ofall Tri-ON oligonucleotides. The product-containing fractions were drieddown in vacuum (SPD 1010 SpeedVac, TermoSavant) and trityl groups wereremoved by treatment in 80% aqueous acetic acid at room temperature for40-60 min. After addition to the detritylation reaction (100 μl) of 20μl sodium acetate (3 M), the oligonucleotide components wereprecipitated in alcohol (1.5 ml), centrifuged, washed with alcohol anddried down. Concentration of the oligonucleotide components wasdetermined based on the optical density at 260 nm and the extinctioncoefficients calculated for individual oligonucleotides using on-lineOligoAnalyzer 3.0 software provided by Integrated DNA Technologies.Based on the measurements, convenient stock solutions in water wereprepared and stored at −20° C. for further use. The purity of allprepared oligonucleotide components was confirmed by analytical 8-20%PAAG electrophoresis, reverse phase HPLC and by spectroscopy on Cary4000 UV-VIS spectrophotometer equipped with Cary WinUV software, BioPackage 3.0 (Varian, Inc.).

Example 2 Synthesis of Circular Oligonucleotide Aptamers

Exemplary circular stem-loop aptamers SEQ ID NOS:6-15 were prepared byligation of the corresponding 5′-phosphate incorporatingoligonucleotides shown in FIGS. 2 and 4 using T4 DNA Ligase kit from NewEngland Biolabs (Catalog number: M0202M). The reaction mixtures wereprepared by mixing 67 μl of 10× ligation buffer and 2.5 μl of T4 DNAligase (2,000 U/μl) from the kit with 10 optical units (at 260 nm) of a5′-phosphate-labelled oligonucleotide (FIGS. 2 and 4) and deionizedwater to provide 670 μl of the final reaction volume. The reactionmixtures were left at room temperature for 1 hour and then heated to 65°C. for 15 min. According to HPLC analysis, the ligation reactions werenearly quantitative (−95%), and the circular aptamers were isolated byHPLC chromatography as described in Example 1. The collected fractionswere dried down, and the circular aptamers were dissolved in water.Concentration of the circular aptamers was determined based on theoptical density at 260 nm as also described in Example 1. Based on themeasurements, convenient stock solutions in water were prepared andstored at −20° C. for further use.

Example 3 Application of Deoxyinosine-Containing Circular Aptamers toControl Polymerase Activity of Taq Polymerase

This working example shows application of deoxyinosine-containingcircular hairpin-like aptamers to control activity of Taq polymeraseduring PCR.

For the results shown in FIGS. 3 and 5, reaction mixtures (25 μL) wereprepared on ice by mixing corresponding stock solutions to provide 200nM forward primer (FIG. 1, SEQ ID NO: 1), 300 nM reverse primer (SEQ IDNO:2), 200 nM FRET probe (SEQ ID NO:3), 0.02 U/μL Taq DNA polymerase(GenScript cat no: E00007), dNTPs (200 μM each), bovine serum albumin(0.1 μg/μL), 100 ng of human genomic DNA (GenScript cat no: M00094) and,when present, one of the aptamers SEQ ID NOS:5-15 (80 nM) in 5 mM MgCl₂,50 mM KCl, 20 mM Tris-HCl (pH8.0). The reaction tubes were quicklytransferred into SmartCycler instrument (Cepheid Corporation) andtemperature cycling initiated. The PCR time/temperature profilecomprised initial incubation at 95° C. for 15 seconds followed by 50cycles of incubation at 95° C. for 1 second and then at 60° C. for 20seconds. The reaction fluorescence was measured in every PCR cycleduring the annealing/extension stage (60° C.) and the results are shownin FIGS. 3 and 5. Each fluorescence curve is an average of fouridentical reactions. Initial background fluorescence was subtracted bythe instrument software.

The reaction conditions used to generate the fluorescence profiles shownin FIGS. 3 and 5 were identical except for the presence or absence ofendonuclease V enzymatic activity and the presence or absence ofdifferent oligonucleotides as potential inhibitors of polymeraseactivity. PCR reactions were performed either in the absence (leftdiagram of each figure) or presence (right diagram) of T. maritimaendonuclease V (0.04 U/μL, Fisher Scientific cat no: FEREN0141) in thereaction mixtures. Structures of the oligonucleotide aptamers used inthe experiments of FIGS. 3 and 5 are shown in FIGS. 2 and 4,respectively.

In summary of this working Example, the real-time curves shown in FIG. 3show that unmodified noncircular aptamer SEQ ID NO:5 has very little, ifany polymerase-deactivating capability whereas its circular analog SEQID NO:6 effectively blocks Taq polymerase activity during PCR, which isnot affected by the presence of endonuclease V activity in the reactionmixture. This result illustrates the effectiveness of aptamercircularization for the deactivation of Taq DNA polymerase. Analysis ofthe real-time PCR curves in FIG. 3 leads to the following conclusions.Virtually identical and very effective results of DNA polymeraseinactivation-activation were obtained for the aptamers SEQ ID NOS:7 and8 incorporating deoxyinosine in the 5′-3′ duplex strand (the strandassignment is shown on the circular aptamer SEQ ID NO:6 of FIG. 2),whereas the results of the deoxyinosine incorporation into the 3′-5′duplex strand of aptamers SEQ ID NOS:9-11 varied significantly. Forexample, circular aptamer SEQ ID NO:9 was very effective at DNApolymerase deactivation (FIG. 3A), but appeared to be refractory to DNApolymerase activation by endonuclease V (FIG. 3B). A likely explanationof these results is that the stem cleavage product of this circularaptamer (SEQ ID NO:9) functions as a self-priming hairpin; that is, thecleaved stem functions like the self-priming fluorescent probe (SEQ IDNO: 16 of FIG. 6) used herein in the exemplary assays to detect andmeasure DNA polymerase activity (FIGS. 7A through 7F). Although thecleaved stem of the circular aptamer (SEQ ID NO:9) is very A-T rich, itis 10 base pairs long and therefore would be relatively stable at 60° C.Extension of this stem cleavage product by DNA polymerase likelycompetes against targeted amplification of the β2-microglobulin sequence(FIG. 1) by the DNA polymerase, resulting in apparent, but not actual,refractory reactivation due to an effective reduction in the rate oftargeted amplification by the DNA polymerase, rather than the circularaptamer SEQ ID NO:9 being refractory to cleavage by endonuclease V.Positioning deoxyinosine closer to the 5′-GAA-3′ loop in circularaptamers SEQ ID NOS:10 and 11 destabilized the stem of the cleavageproduct, eliminating the self-priming problem. Activity of the DNApolymerase, initially blocked by these aptamers, was completely restoredin the presence of endonuclease V (FIG. 3B), but these aptamers wereless effective in initial DNA polymerase inactivation (FIG. 3A), withcircular aptamer SEQ ID NO: 10 performing the worst in this regard. Inconclusion, preferred locations of deoxyinosine substitutions within thestem segment identified herein for highly homologous circularhairpin-like aptamers SEQ ID NOS:6-15 can be design-specific and maychange depending on the stem length, base pair composition (e.g., G-Crichness of duplexes) and particular sequences, etc.

Regarding ability of the circular hairpin-like aptamers to inactivateTaq DNA polymerase, the loop sequence 5′TTCTTAGCGTTT3′ (SEQ ID NO:18) isknown to be highly conserved (e.g., Yakimovich, O. Yu., et al. (2003),Jayasena. S. D. (1999), and U.S. Pat. No. 5,693,502 to Gold, L. andJayasena, S. D.). Nevertheless, a number of the nucleotide substitutionsby deoxyinosine in the loop of circular aptamers SEQ ID NOS:12-15 shownin FIG. 4 were investigated, and the real-time PCR results are providedin FIG. 5. Significant differences in properties were found among thesefour aptamers. For example, the circular aptamer SEQ ID NO: 15 was veryineffective in deactivation of Taq DNA polymerase. In contrast, thecircular aptamer SEQ ID NO: 14 was highly effective for polymerasedeactivation (FIG. 5A), but failed to provide complete EndonucleaseV-mediated reactivation (FIG. 5B); that is, circular aptamer SEQ ID NO:14 provided only partial enzyme reactivation. While not being bound bymechanism, this observation could reflect a reduced rate of cleavage byEndonuclease V at that particular deoxyinosine location within the loop,perhaps due to loop structural constraints. The aptamers SEQ ID NOS:14and 15 represent a homologous guanine-to-hypoxanthine purinesubstitution. The most surprising results were obtained for the circularaptamers SEQ ID NOS:12 and 13 comprising nonhomologous(pyrimidine-purine) thymine-hypoxanthine base alterations within thehighly conservative loop sequence. Both aptamers inactivated the DNApolymerase (FIG. 5A), although the aptamer SEQ ID NO:13 was a much lesseffective inhibitor than the aptamer SEQ ID NO: 12, which was veryeffective in both deactivation (Figure SA) and the EndonucleaseV-mediated activation (FIG. 5B) tests. In conclusion, investigation ofthe deoxyinosine substitutions within the aptamer loop sequencesprovided for identification of at least three exemplary nucleotidelocations that can be used in embodiments of the present invention. Forexample, the loop sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23),5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:25) incorporating 1, 2, or 3 deoxyinosine nucleotides can be used indesign of the circular hairpin-like aptamers for the methods of thepresent invention.

Example 4 Kinetics of Activation by Endonuclease V of Various DNAPolymerases Initially Blocked by Deoxyinosine-Containing Aptamer

This working example shows the kinetics of activation by endonuclease Vof Taq (GenScript cat no: E00007), Q5® (New England Biolabs cat no:M0491S), Vent® (New England Biolabs cat no: M0254S). Deep Vent® (NewEngland Biolabs cat no: M0258S), Bst large fragment (New England Biolabscat no: M0275S), and Phusion® (New England Biolabs cat no: M0530S) DNApolymerases initially blocked by a deoxyinosine-containing aptamers.

For FIG. 7, reaction mixtures (25 μL) were prepared on ice by mixingcorresponding stock solutions to provide self-priming hairpin SEQ IDNO:16 (200 nM, FIG. 8), a DNA polymerase (0.008 U/μL), dNTPs (200 μMeach), bovine serum albumin (0.1 μg/μL) and, when present, the aptamerSEQ ID NO:12 (80 nM, FIG. 4) and T. maritima endonuclease V (0.02 U/μL,Fisher Scientific cat no: FEREN0141) in 5 mM MgCl₂, 50 mM KCl, 20 mMTris-HCl (pH8.0). During preparation of the reaction mixture, theself-priming hairpin (SEQ ID NO: 16) and endonuclease V were alwaysadded last to a premixed solution. Then the reaction tubes weretransferred into a SmartCycler instrument (Cepheid Corporation) andheated to 60 or 65° C. as indicated for each fluorescence profile inFIG. 7. The reaction fluorescence was monitored every 7 seconds. Theplotted curves are the averages of four paralleled identical reactions.Initial background fluorescence was subtracted.

The results of FIG. 7 show that not only Taq polymerase, but also manyother DNA polymerases can be inactivated and then activated usingendonuclease V-cleavable circular aptamers of the present invention.Only one of six investigated exemplary DNA polymerases, particularly BstDNA polymerase, was not inactivated by circular aptamer SEQ ID NO:12.Other DNA polymerases showed an inactivation in the presence of thisaptamer as well as gradual endonuclease V-induced activation, althoughthe efficiency of both processes was variable among the individual DNApolymerases.

REFERENCES CITED, AND INCORPORATED BY REFERENCE HEREIN FOR THEIRRESPECTIVE TEACHINGS

-   Cleuziat, P., and Mandrand, B., “Method for amplifying nucleic acid    sequences by strand displacement using DNA/RNA chimeric    primers,” 1998. U.S. Pat. No. 5,824,517.-   Dattagupta, N., Stull, P. D., Spingola, M., and Kacian, D. L.,    “Isothermal strand displacement nucleic acid amplification,” 2001,    U.S. Pat. No. 6,214,587.-   Davey, C., and Malek, L. T., “Nucleic acid amplification process,”    2000, U.S. Pat. No. 6,063,603.-   Eckstein, F., ed., (1991) Oligonucleotides and Analogs: A Practical    Approach. Oxford University Press, New York.-   Hedgpeth, J., Afonina. I. A., Kutavin, I. V., Lukhtanov, E. A.,    Belousov, E. S., and Meyer, Jr., R. B., “Hybridization and mismatch    discrimination using oligonucleotides conjugated to minor groove    binders,” 2010, U.S. Pat. No. 7,794,945.-   Gait, M. J., ed., (1984) Oligonucleotide Synthesis: A Practical    Approach, IRL Practical Approach Series, IRL Press, Oxford.-   Gold, L. and Jayasena, S. D., “Nucleic acid ligand inhibitors to DNA    polymerase,” 1997, U.S. Pat. No. 5,693,502.-   Jayasena, S. D., “Aptamers: An emerging class of molecules that    rival antibodies in diagnostics,” Clinical Chemistry 45:1628-1650,    1999.-   Komberg, A., and Baker. T. (1992) DNA Replication, Second    Edition, W. H. Freeman and Company, New York.-   Kum, N., “Methods and compositions for linear isothermal    amplification of polynucleotide sequences, using a RNA-DNA composite    primer,” 2001, U.S. Pat. No. 6,251,639.-   Kutyavin, I. V., and Lukhtanov, E. A., Gamper, H. B., Meyer, Jr., R.    B., “Covalently linked oligonucleotide minor grove binder    conjugates,” 1998, U.S. Pat. No. 5,801,155.-   Kutyavin, I. V., “Accelerated cascade amplification (ACA) of nucleic    acids comprising strand and sequence specific DNA nicking,” 2012,    U.S. Pat. No. 8,143,006.-   Kutyavin, I. V., “Use of base-modified deoxynucleoside triphosphates    to improve nucleic acid detection,” 2013, U.S. Pat. No. 8,349,556.-   Lehninger, A. L. (1975) Biochemistry, 2nd edition. New York, Worth    Publishers, Inc.-   Lizardi, P., “Rolling circle replication reporter systems,” 1998,    U.S. Pat. No. 5,854,033.-   Martin, F. H., and Castro, M. M., “Base pairing involving    deoxyinosine: implications for probe design,” Nucleic Acids Res.    13:8927-8938, 1985.-   Meyer, Jr., R. B., Afonina, I. A., and Kutyavin, I. V.,    “Oligonucleotides containing pyrazolo[3,4-D]pyrimidines for    hybridization and mismatch discrimination,” 2000, U.S. Pat. No.    6,127,121.-   Mullis, K. B., “Process for amplifying nucleic acid sequences.”    1987, U.S. Pat. No. 4,683,202.-   Mullis, K. B., Erlich, H. A., Arnheim, N., Horn, G. T., Saiki, R.    K., and Scharf, S. J., “Process for amplifying, detecting,    and/or-cloning nucleic acid sequences,” 1987, U.S. Pat. No.    4,683,195.-   Notomi, T., and Hase, T., “Process for synthesizing nucleic acid,”    2002, U.S. Pat. No. 6,410,278.-   Paul, N., Shum, J., and Le, T. (2010), Hot start PCR. In King N.    (ed.), RT-PCR Protocols: Second Edition. Methods in Molecular    Biology, Springer Science+Business Media, LLC, V.630:301-318.-   Sambrook, J., et al. (1989), Molecular Cloning: A Laboratory Manual,    2nd Edition. Cold Spring Harbor Lab. Cold Spring Harbor, N.Y.-   Walker, G. T., Little, M. C., and Nadeau, J. G., “Nucleic acid    target generation,” 1993, U.S. Pat. No. 5,270,184.-   Yakimovich, O. Yu., Alekseev, Ya. I., Maksimenko, A. V.,    Voronina, O. L., and Lunin, V. G., “Influence of DNA aptamer    structure on the specificity of Binding to Taq DNA polymerase,”    Biochemistry (Moscow) 68:228-235, 2003.-   Yao. M., and Kow, Y. W., “Further Characterization of Escherichia    coli Endonuclease V. Mechanism of Recognition for Deoxyinosine,    Deoxyuridine, and Base Mismatches in DNA,” J. Biol. Chem.    272:30774-30779, 1997.-   Yoshizawa, S., Ueda. T., Ishido, Y., Miura, K., Watanabe, K., and    Hirao, I., “Nuclease resistance of an extraordinarily thermostable    mini-hairpin DNA fragment, d(GCGAAGC) and its application to in    vitro protein synthesis,” Nucleic Acids Res. 22:2217-2221, 1994.

The invention claimed is:
 1. A method of activating anaptamer-inactivated DNA polymerase, comprising: providing a reactionmixture suitable for DNA synthesis, the reaction mixture comprising (i)a DNA polymerase, (ii) an endonuclease V-cleavable circularoligonucleotide aptamer that binds to the DNA polymerase, wherein theoligonucleotide aptamer is present in an amount effective to inhibit DNAsynthesis activity of the DNA polymerase in the reaction mixture, and(iii) an endonuclease V enzymatic activity; and cleaving the aptamer bythe endonuclease V enzymatic activity to reduce or eliminate binding ofthe oligonucleotide aptamer to the DNA polymerase, thereby activatingthe DNA synthesis activity of the DNA polymerase, to increase DNAsynthesis in the reaction mixture.
 2. The method of claim 1, whereinsaid cleaving is facilitated using a reaction temperature thatfacilitates both DNA polymerase activity and the endonuclease Venzymatic activity.
 3. The method of claim 1, wherein said cleaving isfacilitated by increasing the temperature of the reaction mixture from afirst temperature to a second temperature that more strongly facilitatesthe endonuclease V enzymatic activity.
 4. The method of claim 1, whereinsaid providing comprises dissolving a dried form of at least one of said(i) DNA polymerase, (ii) endonuclease V-cleavable circularoligonucleotide aptamer, and (iii) endonuclease V enzymatic activityinto an aqueous solution.
 5. The method of claim 1, wherein the DNAsynthesis results in DNA amplification in the reaction mixture.
 6. Themethod of claim 5, wherein the DNA amplification is an isothermalamplification reaction.
 7. The method of claim 5, wherein the DNAamplification comprises PCR.
 8. The method of claim 5, comprisingdetecting the presence of a target DNA in the reaction mixture.
 9. Themethod of claim 5, comprising measuring an amount of a target DNA in thereaction mixture.
 10. The method of claim 1, wherein the circularoligonucleotide aptamer comprises one or more deoxyinosine nucleotides.11. The method of claim 10, wherein the circular oligonucleotide aptamerhas a stem-loop structure.
 12. The method of claim 11, wherein the oneor more deoxyinosine nucleotides are incorporated into the stem segmentof the stem-loop structure.
 13. The method of claim 11, wherein the oneor more deoxyinosine nucleotides are incorporated into the loop segmentof the stem-loop structure.
 14. The method of claim 13, wherein the loopof the stem-loop structure, comprises a nucleotide sequence selectedfrom the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19),5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21),5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23),5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ IDNO:25).
 15. The method of claim 14, wherein the loop of the stem-loopstructure comprises one of the nucleotide sequences 5′-TTCITAGCGTTT-3′(SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), or 5′-TTCTTAICGTTT-3′(SEQ ID NO:21).
 16. The method of claim 1, wherein the endonuclease Venzymatic activity comprises Thermotoga maritima endonuclease Venzymatic activity.